Busulphan (1,4-bis-(methanesulphonyloxy)butan) is a bifunctional alkylating agent with potent antitumor effects. It has been widely used for treatment of malignant diseases, especially hematological malignancies and myeloproliferative disorders. Its use was for a long time restricted to low dose oral therapy, with recorded side effects such as busulphan-induced pulmonary fibrosis (Oakhill et al. 1981. J. Clin. Pathol. 34(5):495-500.) and irreversible myelo-suppression (Canellos 1985. Chronic Leukemias In: Cancer: Principles and Practice of Oncology, pp 1739-1752). In 1983, high-dose combination chemotherapy based on oral busulphan for pretransplant-conditioning of patients undergoing both autologous and allogeneic bone marrow transplantation (Santos, G. W. et al. 1983. N. Engl. J. Med. 309: 1347-1353; Lu, C. et al. 1984. Cancer Treatm. Repts. 68: 711-717) was introduced. Since then, high dose busulphan replaces total body irradiation (TBI), most commonly in combination with cyclophosphamide, and has proven to be a most effective anti-leukemic regimen when used in conjunction with autologous or allogeneic hematopoietic stem cell support. The main advantage of high-dose busulphan therapy for use in marrow ablation treatment is that total body irradiation regimen is avoided, which is especially advantageous for young children and adults who received TBI as part of their initial therapy.
Oral busulphan in combination with cyclophosamide has several serious side effects. Reported side effects are fatal liver failure (Miller, C. et al. 1991. Blood 78: 1155), neurological disturbances like grand mal seizures, veno-occlusive disease, severe nausea and vomiting (Vassal, G. et al. 1990. Cancer Res. 50: 6203-6207) as well as side effects in the lungs, such as interstitial pneumonia (Bandini, G. et al. 1994. Bone Marrow Transpl. 13: 577-581).
Therefore attempts have been made to improve the clinical utility of the busulphan drug by providing a busulphan formulation for parenteral administration. U.S. Pat. Nos. 5,430,057 and 5,559,148 disclose physiologically acceptable solutions of busulphan, wherein N′,N-dimethyl-acetamide, polyethylene glycol, propylene glycol, glycerin cyclodextrin, hydroxypropylbetacyclodextrin are used as solvents. In two other studies busulphan was dissolved in dimethylacetamide (DMA) (Bhagwatwar H. P, Phadungpojna S, Chow D. S., Andersson B. S. 1996. Cancer Chemother Pharmacol 37: 401) and dimethylsulfoxide (DMSO) (Ehninger G, Schuler U, Renner U, Ehrsam M, Zeller K. P, Blanz J, Storb R, Deeg H. J. 1995. Blood 85: 3247). However, despite the very promising results, these organic solvents have their own toxicity (Kennedy Jr G. L, Sherman H. 1986. Drug and Chemical Toxicology 9: 147; Kinney L. A, Burgess B. A, Stula E. F, Kennedy Jr G. L. 1993. Drug and Chemical Toxicology 16: 175; Malley L. A, Slone Jr T. W, Makovec G. T, Elliott G. S, Kennedy Jr G. L. 1995. Fundam Appl Toxicol 28: 80; Martino M, Morabito F, Messina G, Irrera G, Pucci G, Iacopino P. 1996. Haematologica 81: 59; Sperling S, Larsen I. G. 1979. Acta Opthalmol (Copenh) 57: 891; Yellowlees P, Greenfield C, McIntyre N. 1980. Lancet 2: 1004). Further, it seems as if DMSO activates the metabolism of busulphan in the liver, resulting in reduced concentrations of busulphan in the blood of the patient after a few days regimen (Shuler et al. 1997. Abstract EBMT-meeting, Chamonix, France). The Spartaject™ technology has tried to achieve a formulation of busulphan for parenteral administration by forming microcrystals of busulphan coated with lecithin (or other phospholipid) dissolved in mannitol and water. The size of these microcrystals is 0.1 μm. The main problem with this formulation is precipitation of the microcrystals during infusion of the drug. Also nothing is known about their biodistribution. Both busulphan dissolved in organic solvents and oral busulphan cross the blood brain barrier, giving rise to central nerve system toxicity.
Several studies have shown a correlation between exposure to busulphan and transplantation related liver toxicity, such as venoocclusive disease (VOD) in patients undergoing SCT (Ringdén O, Ruutu T, Remberger M, et al. A randomised trial comparing busulfan with total body irradiation as conditioning in allogeneic marrow transplant recipients with leukemia: A report from the Nordic Bone Marrow Transplant Group. Blood 1994; 83: 2723-2730; Grochow L B, Jones R J, Brundrett R B, Braine H G, Chen T L, Saral R, et al. Pharmacokinetics of busulfan: correlation with venoocclusive disease in patients undergoing bone marrow transplantation. Cancer Chemother Pharmacol 1989; 25(1): 55-61; Bearman S I. The syndrome of hepatic venoocclusive disease after marrow transplantation. Blood 1995; 85: 3005; Shulman H M, Hinterberger W. Hepatic venoocclusive disease—liver toxicity syndrome after bone marrow transplantation. Transplantation 1992; 10: 197.). Apart from acute GVHD and infections, VOD is one of the common early complications with a potential fatal outcome following SCT. Hepatic VOD is a clinical syndrome consisting of jaundice, ascites and/or unexplained weight gain, and hepatomegaly and/or upper quadrant abdominal pain. It is a life-threatening complication and the reported incidences vary considerably; though it may improve spontaneously, the mortality of severe VOD is about 20-50%.
Another problem of the prior art methods for treatment with busulphan as a part of the myeloablative regimen prior to stem cell transplantation is that the dosage has been difficult to optimize due to the wide inter-patient variability in pharmacokinetics in combination with the narrow therapeutic window of busulphan. A suboptimal dosage of busulphan expressed as low AUC after oral administration was correlated to a minimal toxicity in children and both graft rejection and relapse in adults (Slattery J T, Sanders J E, Buckner C D, et al: Graft-rejection and toxicity following bone marrow transplantation in relation to busulfan pharmacokinetics [published erratum appears in Bone Marrow Transplant 1996 October; 18(4):829]. Bone Marrow Transplant 16: 31-42, 1995; Pawlowska A B, Blazar B R, Angelucci E, et al: Relationship of plasma pharmacokinetics of high-dose oral busulfan to the outcome of allogeneic bone marrow transplantation in children with thalassemia. Bone Marrow Transplant 20: 915-20, 1997). On the other hand, as mentioned herein above, high exposure to busuphan during conditioning has been correlated to VO and also interstitial pneumonia and CNS toxicity (Vassal G, Deroussent A, Hartmann O, et al: Dose-dependent neurotoxicity of high-dose busulfan in children: a clinical and pharmacological study. Cancer Res 50: 6203-7, 1990; Grochow L B, Jones R J, Brundrett R B, et al: Pharmacokinetics of busulfan: correlation with veno-occlusive disease in patients undergoing bone marrow transplantation. Cancer Chemother Pharmacol 25: 55-61, 1989; Dix S P, Wingard J R, Mullins R E, et al: Association of busulfan area under the curve with veno-occlusive disease following BMT. Bone Marrow Transplant 17: 225-30, 1996). High plasma concentrations of busulphan were also correlated to transplantation related mortality occurring before day 100 post transplantation (Ljungman P, Hassan M, Bekassy A N, et al: High busulfan concentrations are associated with increased transplant-related mortality in allogeneic bone marrow transplant patients. Bone Marrow Transplant 20: 909-13, 1997). In a randomized study, busulphan compared to total body irradiation has been found to be associated with an increased risk of hemorrhagic cystitis, VOD, obstructive bronchiolitis, alopecia and chronic GVHD (Ringden O, Remberger M, Ruutu T, et al: Increased risk of chronic graft-versus-host disease, obstructive bronchiolitis, and alopecia with busulfan versus total body irradiation: long-term results of a randomized trial in allogeneic marrow recipients with leukemia. Nordic Bone Marrow Transplantation Group. Blood 93: 2196-201, 1999).
To optimize treatment with oral busulphan and to minimize its toxicity during high dose therapy, many investigators have suggested therapeutic monitoring using limited sampling models followed by dose adjustment (Vassal G, Deroussent A, Challine D, et al: Is 600 mg/m2 the appropriate dosage of busulfan in children undergoing bone marrow transplantation? Blood 79: 2475-9, 1992; Hassan M, Fasth A, Gerritsen B, et al: Busulphan kinetics and limited sampling model in children with leukemia and inherited disorders. Bone Marrow Transplant 18: 843-50, 1996; Chattergoon D S, Saunders E F, Klein J, et al: An improved limited sampling method for individualised busulphan dosing in bone marrow transplantation in children. Bone Marrow Transplant 20: 347-54, 1997; Schuler U, Schroer S, Kuhnle A, et al: Busulfan pharmacokinetics in bone marrow transplant patients: is drug monitoring warranted? Bone Marrow Transplant 14: 759-65, 1994). However, the usefulness of this strategy is limited due to the restricted possibility to perform sample analysis, the losses of the drug through emesis and/or the irregular and slow absorption reported in some patients.
From the above, it appears that treatment of a mammalian patient with busulphan suffers from serious drawbacks due to the chemical nature of the busulphan molecule that makes its administration and proper dosage problematic, as well as to the inherent toxicity of the molecule, resulting in secondary effects that may lead to life-threatening conditions.
The tripeptide glutathione (g-glutamylcysteinylglycine, GSH) is an important component of the intracellular defense against toxic challenge. It is a sulfhydryl (—SH) antioxidant, an antitoxin, and enzyme cofactor, ubiquitous in animals, plants, and microorganisms, often attaining millimolar levels inside cells.
It is believed that low constitutive levels of GSH in centrilobular hepatocytes, together with further GSH exhaustion by cytotoxic drugs or radiation contributes to development of VOD (Carreras E. Venoocclusive disease of the liver after hematopoietic cell transplantation. Eur J Haematol 2000; 64(5): 281-91). Involvement of GSH in the metabolism of busulphan has been studied in animal models and in man (Marchand D H, Remmel R P, Abdel-Monem M M. Biliary excretion of a glutathione conjugate of busulfan and 1,4 diiodobutane in the rat. Drug Metab Dispos 1988; 16(1): 85-92; Hassan M, Ehrsson H. Metabolism of 14C-busulfan in isolated perfused rat liver. Eur J Drug Metab Pharmacokinet 1987; 12(1):71-6; Ritter C A, Bohnenstengel F, Hofmann Um Kroemer H K, Sperker B. Determination of tetrahydrothiophene formation as a probe of in vitro busulfan metabolism by human glutathione S-transferase A1-1: use of a highly sensitive gas chromatographic-mass spectrometric method. J Chromatogr B Biomed Sci Appl 1999; 730(1):25-31). Several investigations have conformed that busulphan is metabolized in the liver through conjugation with GSH catalyzed by glutathione S-transferase (GST). The reaction is catalyzed in human liver mainly by GST-A1-1 (Czerwinski M, Gibbs J P, Slattery J T. Busulfan conjugation by glutathione S-transferase alpha, my and pi. Drug Metab Dispos 1996; 24(9): 1015-9; Gibbs J P, Czerwinski M, Slattery J T. Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases. Cancer Res 1996; 56(16):3678-81). Treatment with busulphan depleted GSH by 60% in murine hepatocytes in vivo and by 50% in vitro (De Leve L D, Wang X. Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes. Pharmacology 200; 60(3):143-54). In the same study, modulation of the cellular levels of GSH changed hepatocyte cytotoxity of busulphan: cells depleted of GSH were more sensitive and cells with increased GSH less sensitive to the toxic effect. Precursors of GSH, such as N-acetyl-L-cysteine (NAC) or methionine, increase cellular content of GSH (Meister A. Glutathione metabolism and its selective modification. J Biol Chem 1988; 263(33):17205-8).
The GSH precursor NAC is used to treat hepatotoxicity induced by acetaminophen (Chyka, P. A., Butler, A. Y., Holliman, B. J., and Herman, M. I. Utility of acetylcysteine in treating poisonings and adverse drug reactions. Drug Saf, 22: 123-148, 2000.). NAC has few and mild side effects. Recently, a case report on three patients with VOD, who were successfully treated with NAC, was published (Ringden, O., Remberger, M., Lehmann, S., Hentschke, P., Mattsson, J., Klaesson, S., Aschan, J.: N-acetylcysteine for hepatic veno-occlusive disease after allogeneic stem cell transplantation. Bone Marrow Transplant. 25: 993-996, 2000).
On the other hand, depletion of GSH increases toxicity of alkylating agents in most cell systems studied. GSH may be depleted in vivo by treatment with buthionine sulfoximine and in vitro by incubation in medium without sulphur amino acids, or by treatment with buthionine sulfoximine or ethacrynic acid (Mulder, G. J. and Ouwerkerk-Mahadevan, S. Modulation of glutathione conjugation in vivo: how to decrease glutathione conjugation in vivo or in intact cellular systems in vitro. Chem Biol Interact, 105: 17-34, 1997.). L-buthionine-[S,R]-sulfoximine (BSO) is an irreversible inhibitor of γ-glutamylcysteine synthetase, an enzyme catalyzing the first step of de novo synthesis of GSH in cells (Griffith, O. W. and Meister, A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem, 254: 7558-7560., 1979.).
Prior to the present invention, the effect of modulation of GSH on busulphan-induced cytotoxicity had not been studied in hematopoietic cells. Though hematopoietic stem cells do not express GSTA1, they do express other GST isoforms. In analogy with the cytoprotective effect obtained by administration of a GSH precursor to hepatocytes, a reasonable hypothesis would be that such administration would also help protecting target cells, such as hematopoietic cells during a myealoablative treatment, against the cytotoxic effects of an alkylating agent such as busulphan, thereby reducing the treatment efficiency. This would seem to rule out use of GSH or a GSH precursor during busulphan treatment.
A liposome is a completely closed lipid bilayer membrane which defines a closed aqueous compartment. Liposomes are microscopic delivery vesicles made, in part, from phospholipids which form closed, fluid filled spheres when mixed with water. Phospholipid molecules are polar, having a hydrophilic ionizable head, and a hydrophobic tail consisting of long fatty acid chains. Thus when sufficient phospholipid molecules are present with water, the tails spontaneously associate to exclude water while the hydrophilic phosphate heads interact with water. Liposomes may be either unilamellar, comprised of one lipid bilayer membrane, or bilayer liposomes, comprised of two layers of lipids. In the latter liposomes the outer layer of lipid molecules are oriented with their hydrophilic head portions towards the external aqueous medium and their hydrophobic tails pointed inwards towards the interior of the liposome. The inner layer of lipids lies directly beneath the outer layer; the lipids are oriented with their heads facing the aqueous interior of the liposome and their tails towards the tails of the outer lipid layer. Multilamellar liposomes are composed of more than one lipid bilayer membrane, which define more than one closed compartment and are concentrically arranged.
Since the chemical composition of many drugs precludes their intravenous administration, liposomes can be very useful in adapting these drugs for intravenous delivery. Many hydrofobic drugs fall into this category because they cannot be easily dissolved in a water-based medium and must be dissolved in alcohols or surfactants which have been shown to cause toxic reactions in vivo. Liposomes, composed of predominantly lipids, with or without cholesterol, are nontoxic. Further, liposomes have the potential of providing a controlled release of the administered drug over an extended period of time, and of reducing toxic side effects of the drug, by limiting the concentration of the free drug in the bloodstream. Liposomes can also alter the tissue distribution and uptake of drugs, and the altered tissue distribution can significantly increase the therapeutic effectiveness of the drug. Liposome/drug compositions can for these reasons increase the convenience of therapy by allowing higher drug dosage and less frequent drug administration.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 13238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film or powder. Next, hydration is performed, i.e. an aqueous phase is added to the phospholipid film and the mixture is dispersed by mechanical means resulting in liposomes consisting of multilamellar vesicles (MLVs). This preparation provides the basis for the development of small sonicated unilamellar vesicles (SUVs) described by Papahadjopoulos et al. (Biochim. Biophys. Acta. 1967, 135: 624-638), and large unilamellar vesicles (LUVs).
A variety of sterols and their water soluble derivatives such as cholesterol hemisuccinate as well as a variety of tocopherols and their water soluble derivatives have been used to form liposomes. Liposomes themselves have been reported to have no significant toxicities in previous human clinical trials where they have been given intravenously.
Prior to the present invention, busulphan had been therapeutically used for a very long time and at the priority date of the application the toxicity of busulphan was well-known. However, in spite of the serious and well-documented side-effects of busulphan therapy and in spite of the knowledge that the toxicity might be reduced by liposomal encapsulation of busulphan, prior to the application this had not been practically done and there existed no practical method permitting to do this.
In the literature (Weiner et al: Lipsomes as a Drug Deliver System, Drug Development and Industrial Pharmacy, 15(10), 1523-1554), it has been pointed out that “the maximum amount of drug that can be entrapped within a liposome is dependent on its total solubility in each phase”. Busulphan has very limited solubility in both polar and non-polar solvents. Though described as a lipophilic compound, it in fact is poorly soluble in oils, carbon tetrachloride or any other very lipophilic solvent. Busulphan also is only very slightly water soluble (less than 1 microgram/ml at 25° C.). Therefore, prior to the present invention there existed no way of preparing a liposomal composition having therapeutically useful content of busulphan enclosed in liposomes.